Troponin I, the inhibitory subunit of troponin, is an important regulatory protein in striated muscle. Separate genes encode three tissue-specific isoforms of this protein. In mature fast- and slow-twitch skeletal and cardiac muscle these isoforms are specifically expressed, however, in fetal and neonatal rat and human heart the cardiac and slow skeletal troponin I mRNAs and isoforms are co-expressed. Alterations in the response of the contractile apparatus to acidosis and beta-adrenergic stimulation may result from the presence of slow skeletal troponin I in the immature heart. The long term objectives of-this work are to understand the functional consequences of troponin I isoform variation in the heart, and to understand the cis- and trans-acting factors which allow expression of the slow skeletal and cardiac troponin I mRNAs in immature heart, and those which direct specific expression of cardiac troponin I in the mature heart. The specific aims of this proposal are: 1. To characterize the regulatory regions of the rat cardiac troponin I gene by the use of transient expression assays of reporter gene constructs transfected into primary neonatal rat cardiocytes. The regions to be characterized include elements which regulate expression during cardiac maturation and elements which direct cardiac-specific expression. 2. To obtain genomic clones of rat slow skeletal troponin I by screening rat genomic libraries in lambda vectors. To characterize regulatory regions of this gene by the use of transient expression assays of reporter gene constructs in skeletal muscle cell lines, and in primary cardiocytes. The elements to be characterized include those which confer muscle-specificity, those which inhibit expression during cardiac maturation, and elements responsive to thyroid hormone. 3. To determine if fast skeletal troponin I or an "embryonic" cardiac troponin I isoform is expressed in embryonic or fetal rat heart, and the time course of its expression. If an embryonic gene is found, to determine whether its mRNA is re-expressed in a model for pressure overload hypertrophy created by banding the aorta of adult rats. 4. To purify recombinantly expressed wild type and mutant rat cardiac troponin I in order to assess regions of the cardiac troponin I molecule which result in the greater sensitivity of the contractile apparatus to acidosis when the cardiac isoform in present in the troponin complex.